Polymeric immunological adjuvants

ABSTRACT

Adjuvants for enhancing the immune response to an antigen are provided comprising the adjuvant incorporated into a lipid layer where the adjuvant is covalently or non-covalently involved in a polymeric system. Conveniently, the adjuvant may be conjugated to a polymerizable group and co-polymerized with a water-soluble and/or amphiphilic polymerizable monomer or combined with a polymerized amphiphile. The adjuvant and antigen may then be administered to a mammalian host to obtain enhanced immune response.

This is a continuation of application Ser. No. 07/144,408 filed Jan. 15,1988 now abandoned.

TECHNICAL FIELD

The field concerns methods and compositions for employing immunizationadjuvants to enhance immune responses. The compositions involve variouslipids, polymers, and/or polypeptides.

BACKGROUND

Adjuvants by definition are substances that are incorporated into or areinjected simultaneously with an antigen. The adjuvants potentiatenonspecifically the ensuing immune response. A principal purpose foremployment of an immunotherapeutic adjuvant is to achieve more durablehumoral or cell-mediated immunity of a high level by employing lowerlevels of an antigen with fewer numbers of doses than could be achievedby administering the equivalent aqueous antigen. Adjuvants are used incombination with non-living agents (in place of living microorganisms),for the preparation of vaccines. Adjuvants may also increase theeffective immune response against low or nonimmunogenic tumor cells orcells infected with intracellular agents that are already present in thebody and are not adequately checked by naturally elicited immuneresponses.

As studies slowly unravel the cellular and molecular mechanismsresponsible for host immune responses, the challenge to design newcompounds necessary to modulate both humoral and cell-mediated immunitybecomes increasingly apparent. While hypotheses have been proposed as tohow adjuvants augment the immune response, no substantial evidenceexists which supports any particular theory or provides for a predictiverationale which can serve as a basis for designing improved adjuvants.To that extent then, while one can extrapolate from adjuvants which havebeen shown to be successful, there is still substantial uncertainty asto whether there will be success with new compositions.

With the advent of genetic engineering, there is now the possibility todevelop any protein molecule, which can be used to mimic an epitope ofan antigen of interest. For the most part, proteins by themselves do notelicit strong immune responses, as compared to a cell containing suchprotein. There is, therefore, an increased interest in being able todevelop adjuvants which will potentiate immune responses to proteins orother antigenic compositions, e.g. saccharide or hapten conjugates, soas to produce neutralizing antibodies or cell-mediated immunityeffective for protecting a host against a pathogen.

Relevant Literature

Ribi, Structure-Function Relationship of Bacterial Adjuvants, In:Advances in Carriers and Adjuvants for Veterinary Biologics, Nervig,Gough, Kaeberle and Whetstone, eds., Iowa State University Press, Ames,Iowa, 1986, p. 35-49, describes improvements in bacterial adjuvants.

Other references of interest concerning adjuvants include Ribi et al.,J. Natl. Cancer Inst. (1975) 55:1253; Ribi, J. Biol. Resp. Mod. (1984)3:1-9; Ribi et al., BCG Cell Wall Skeleton, P3, MDP and Other MicrobialComponents-Structural Activities in Animal Models, In: Augmenting Agentsin Cancer Therapy, Hersh, Chirigos and Mastrangelo, eds., Raven Press,New York, 1981, p. 15ff; Ribi et al., Rev. Infect. Dis. (1984)6:567-572; Takayama et al., Rev. Infect. Dis. (1984) 6:439; Chase etal., Inf. and Immun. (1986) 53:71; Ribi et al., Modulation of Humoraland Cell-Mediated Immune Responses by a Structurally EstablishedNontoxic Lipid A, In: Proc. Symp. on Bacterial Endotoxins, Tampa, Fla.,Jan. 1985; Immunology and Immunopharmacology of Bacterial Endotoxins,Plenum Publishing Inc., NY, 1986, p 407; Ribi et al., ImmunoPotentiating Activities of Monophosphoryl Lipid A, In: Int. Sym. onImmunological Adjuvants and Modulators of Nonspecific Resistance toMicrobial Infections, Columbia, Md., July 1986, Alien Liss Inc., NY,1986; Ribi et al., Enhancement of Tumor Immunity with BacterialAdjuvants, In: Development in Industrial Microbiology, vol. 27, supp. 1,1987, p. 19; Tomai et al., J. Bio. Resp. Mod. (1987) No. 6, 99; andPhilip et al., Cancer Res. (1985) 45:128-134.

References related to the polymerization of lipids and drugs includeRingsdorf, J. Polym. Sci. (1975) Symp. No. 51, 135; Przybylski et al.,Makromol. Chem. (1978) 179:1719; Hirano et al., Tetra. Lett. (1979)10:883; Hirano et al., Cancer Research (1980) 40:2263; Gros et al.,Angew. Chem. Int. Ed. Engl. (1981) 20:305; Kobayashi et al., Makromol.Chem. (1983) 184:793; Bader et al., Angew. Makromol. Chem. (1984)123/124:457; Pratten et al., Makromol. Chem. (1985) 186:725; Elbert etal., J. Am. Chem. Soc. (1985) 107:4143; Sackmann et al., Ber. Bunsenges.Phys. Chem. (1985) 89:1208; Dorn et al., Polymeric Antitumor Agents on aMolecular and Cellular Level, In: Bioactive Polymer Systems, AnOverview, Gebelein and Carraher, eds., Plenum Press, NY, 1985, 19:531;and Matsumura and Takahashi, Makromol. Chem. Rapid Commun. (1986) 7:369.

Gaub et al., Biophys. J. ( 1984) 45:725-731; Laschewsky et al., DieAngewandte Makromolekulare Chemie (1986) 145/146:1-11; Frey et al.,Macromolecules (1987) 20:1312-1321, describe the physical properties ofpolymerized and copolymerized amphiphiles. See also, Gorbach et al.,Bioorganicheskaya Khimiya (1985) 11:671-673.

SUMMARY OF THE INVENTION

Polymeric immunologically active adjuvants and methods of use areprovided, where the adjuvants may be used in immunotherapeuticapplications or production of antibodies. The polymeric adjuvantscomprise a pharmacologically acceptable lipid where the adjuvant iscovalently or non-covalently associated with the polymer. Polymericlipid-adjuvants take the form of lipid layers or colloidal particles.The polymers may be prepared in a variety of ways and the polymericproduct administered separately or in combination with an immunogen.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Immunotherapeutic adjuvants are comprised of polymer-containing lipidlayers which may be present in a colloidal form (e.g. vesicles). Thelipid layers include the adjuvant active component. The adjuvant activecomponent may be the lipid layer forming component, or covalently ornon-covalently associated with the lipid layer forming component.Depending upon the polymer, the adjuvant active compound may becovalently bonded to the polymer or non-covalently associated withhydrophilic or hydrophobic portions of the polymer.

Bacterially derived immune modulators (adjuvants) exist naturally withinthe polymer matrix of bacterial cell walls. Isolated and detoxifiedforms of these adjuvants have been shown to retain many of theirbeneficial properties. Monomeric forms of bacterial adjuvants are,however, unstable and exhibit reduced half lives. Repolymerization ofpurified forms of these adjuvants, thus serves several objectives:first, to reconstitute the adjuvant component into a relevant context, apolymer form; second, to allow for flexibility in the polymericcomposition, in order to vary the physical/chemical properties of thesystem in vivo; and third, to provide for more stable adjuvantformulations (during storage and in vivo).

The polymeric adjuvant composition will comprise at least onepolymerized lipid moiety having at least three monomeric units. Thecompostion will include at least one of a lamellar forming lipidadjuvant and, possibly, a lamellar forming "non-adjuvant" lipid, wherewith the "non-adjuvant" lipid, the adjuvant will be present eitherco-polymerized or linked to a lipid. By "non-adjuvant" is intended alamellar forming lipid which by itself does not enhance immune responseto an antigen. By lamellar forming is intended self-organizing lipidswhich are able to form stable mono- or multilayers in solution or atinterfaces.

Polymeric adjuvant systems serve several purposes: first, to potentiateand regulate beneficial immune responses; second, to provide a highdegree of colloidal stability and half life of the adjuvants both beforeand after injection into the host; and third, to create a controllableadjuvant release system (a depot) which avoids the use of oils which maycause toxic side effects to the host.

The adjuvants for the most part will comprise at least one lipid orpeptide and at least one polyol, usually a sugar, e.g. an aminosugar,such as muramic acid or a disaccharide such as trehalose or glucosaminedisaccharide; or glycerol, e.g. phosphatide, phosphoinositide diglycerolether, etc. Illustrative adjuvants include monophosphoryl lipid A (MPL),diphosphoryl lipid A (DPL), muramyl dipeptide-phosphatidylethanolamine(MDP-PE), muramyl tripeptide-phosphatidylethanolamine (MTP-PE), mycolicacid (e.g. arabinomycolate), trehalose monomycolate, trehalosedimycolate (TDM), lipid X (LX), or other adjuvant forms such asisoprinosines and plant lithospermans.

The polymer and adjuvant-containing compositions involve an adjuvantcovalently bonded to a polymerizable monomer or covalently bonded tolipid. The polymeric compositions will involve adjuvant-lipid conjugatemonomer combined with polymeric lipid; adjuvant-(polymerizable monomer)oligomer combined with polymeric or non-polymeric lipid;adjuvant-(polymerizable monomer) copolymerized with polymerizable lipid;and adjuvant(lipid)-(polymerizable monomer) polymerized with non-lipidmonomer. In all of the compositions, the adjuvant will be flexiblyassociated with the polymer, such as through non-covalent lipidassociation, linkage through a flexible, usually long aliphatic,hydrophilic chain and/or adjuvant-polymerizable monomer conjugatecopolymerized with hydrophilic monomer to provide a significant spacerbetween adjuvant conjugate monomers in the polymer. Thus, either theadjuvant or the lipid may be monomeric or polymeric and where adjuvantis lipophilic, no other lipid need be employed, unless to modify thecomposition.

The polymers may be any convenient oligomer for incorporating the activeadjuvant component. The polymers may be natural or synthetic polymers orcombinations thereof, the polymer usually having at least about 3 unitsand may have 10,000 or more units, the size of the polymer not beingcritical to this invention, but rather associated with ease offormulation and administration, as well as physiological acceptability.The polymers may be biodegradable or non-biodegradable.

The polymers may be polyesters, polyacrylates, polyethers, polypeptides,polyolefins, polyacetylenes, particularly polydiacetylenes, polyenes,polyamides, disulfides, polysilanes and polysilynes, polycyanocompounds, or the like. The polymeric backbone may include suchheteroatoms as oxygen, sulfur, nitrogen, silicon, phosphorus, or thelike.

The polymers may be addition or condensation polymers, usually additionpolymers. Functionalities which may be present include aliphaticunsaturation--olefins or acetylene--esters, both organic and inorganic,ethers, both oxy and thio, amides, hydroxy, amino, nitro, cyano, etc.The polymers may be a backbone polymer with lipid groups pendant fromthe backbone or the polymer may involve polymerization of the lipidgroups, e.g. through polyunsaturation, or the like.

Illustrative polymers include acrylic or methacrylic acid polymers,particularly derivatives thereof, such as amides and esters, both ashomo- or copolymers, having side groups, such as hydroxyethyl,aminopropyl, methyl, ethyl, 2-aminoethyl, 2-ammoniumethyl, or the like;polyvinyl alcohol and esters thereof, e.g. acetate or fatty acids offrom about 8 to 100 carbon atoms, particularly of from about 12 to 30carbon atoms; polydiacetylenes, particularly where the diacetylenes areinternal to aliphatic chains of at least about 8 to 60 carbon atoms,more usually up to about 30 carbon atoms; substituted polybutadiene orpolyisoprene; polysuccinates, employing glycols of from about 2 to 10carbon atoms; polyamides, such as polyadipamide or polysuccinamide witha diamine of from about 2 to 10 carbon atoms where from about 1 to 20mol % of the monomers which do not have functionalities for attachmentmay be substituted for a monomer having a site for attachment, e.g.succinic by maleic or fumaric, propylene glycol by dihydroxyacetone,isoprene by 2-carboxymethylbutadiene, polydisulfides, with thesulfhydryl moieties attached to the aliphatic chains or polar groups,etc.

Self-organization of polymeric adjuvants into stable colloidalstructures may further require co-polymerization of the adjuvant withsmall polymerizable molecules (co-monomers, usually hydrophilic).Co-polymerization allows the spacings between adjuvant molecules, alongany given polymer chain, to be regulated by varying the mol percentageof the co-monomer. Oriented monolayers, multilayers, and vesicles havebeen prepared by this approach Laschewsky, A. et al., supra; Frey, W. etal., supra)

It may be desirable for the hydrophilic-polymerizable co-monomer toinclude a hydrophilic adjuvant such as MDP or MTP derivatized with apolymerizable group (e.g. acryl, methacryl, 2-hydroxethyl-acrylate,etc.). Co-polymerization of a hydrophilic adjuvant with a lipophilicadjuvant, bearing a compatible polymerizable group, provides for theformation of mixed polymeric adjuvants which are both stabilized by thepolymeric backbone and able to self-assemble into colloidal structures.Alternatively, mixed adjuvant systems may be comprised solely of variouslipophilic adjuvants co-polymerized on the same polymer backbone(heteropolymers) or each polymerizable adjuvant may be polymerizedindividually (homopolymers) and then mixed together.

The polymers will have a lipid or hydrophobic group present in a ratioof at least one aliphatic chain of at least 8 carbon atoms per 100monomer units, and there may be 2 or more hydrophobic chains per monomerunit. The number of lipid groups will be selected to provide for astable colloidal particle (lipid layers, including vesicles asappropriate).

The active adjuvant entity may be present as part of the polymer asbeing covalently bonded directly or through a linking group to afunctionality present on the polymer backbone or may be non-covalentlyassociated with the hydrophobic groups of the polymer in the lipidlayer. For covalent linkage of the adjuvant to the polymeric backbone,monomers which do not have a convenient functional group, may bechemically modified to introduce an occasional functional group forlinking the adjuvant to the polymer in an appropriate ratio.

Introducing polymerizable groups into adjuvant molecules involveschemically modifying the adjuvant without perturbing the adjuvant'sbeneficial properties. Polymerizable moieties are introduced throughcovalent attachment to functional groups at various positions on theadjuvant. This can be achieved in a variety of ways, such as providingfor olefinic groups for coupling with a mercaptan, hydroxyl groups toform esters and ethers, amino groups, to provide for amides oralkylation, for example with an aldehyde under reductive aminationconditions. Usually the linking site will be the hydroxyl group of asugar or through an amino group of a sugar or side chain. For separatingthe polymerizable group from the adjuvant and to provide for flexibilityin the polymer chain, spacer groups may be employed. The spacersfacilitate the polymerization process and allow the adjuvant to functionin a relevant context, without interference from the polymer backbone.It is often desirable to attach spacers and polymerizable groups atdifferent positions on the adjuvant, depending upon the chemicalstructure of the adjuvant, type of polymerizable group, polymerstructure of interest, and effect that the polymer backbone may have onthe adjuvant's properties.

The linking group may be any bifunctional group which allows for linkingof the adjuvant to the polymeric backbone. Of particular interest willbe those linking groups which include such functionalities as hydroxyls,halogens, aminos, carboxyls, thiols, phosphoroyls, or the like. Thelinking group will usually have at least one carbon atom, more usuallyat least two carbon atoms and could have 100 carbon atoms or more, thechain being a matter of convenience. Usually, the chain will be 20carbons or fewer, preferably 12 carbons or fewer. The linking group maybe aliphatic, alicyclic, aromatic or heterocyclic, or combinationsthereof. The linking group may be aliphatically saturated orunsaturated, usually having fewer than about two sites of unsaturation,and may be hydrophobic or hydrophilic. Illustrative linking groupsinclude hydroxyamines, such as ethanolamine, saccharides, such asglucose, fructose, ribose, etc., ethylene or polyethylene glycols(oxides) or homologs thereof, e.g. mono- or polypropylene glycols, alkyldiamines such as propylene diamine or 1,6-diaminohexane, dicarboxylicacids such a maleic, fumaric, succinic, adipic, suberic, etc., hydroxyor amino acids or peptides, such as glycolic acid, 6-aminohexanoic acid,di- or triglycine, cysteine, etc., or the like.

The adjuvant monomer may or may not have lipid moieties. Non-lipidadjuvants may be chemically coupled to a lipid to provide for anamphiphilic molecule. The lipid molecules may be conjugated to a varietyof hydrophilic monomers, particularly addition polymerizable monomers.

Illustrative hydrophilic adjuvant monomers, which may be coupled topolymerizable lipids, include various muramyldipeptides (e.g.N-acetylmuramyl-L-threonyl-D-isoglutamine orN-acetylmuramyl-L-alanyl-D-isoglutamine). Hydrophilic adjuvant monomersderivatized with a polymerizable moiety (e.g. N¹-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N⁶-acryloylhexamethylenediamine, or N⁴-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-triglycylacrylamide), maybe coupled to a non-polymerizable lipid.

Illustrative lipophilic adjuvants, modified with polyermizable moietiesinclude lipid A derivatives, such astetraethyleneglycolacrylate-ether-O⁶ -monophosphoryl lipid A, andtrehalose dimycolate derivatives, such asvinylether-tetraethyleneglycolether-trehalose dimycolate.

Illustrative polymerizable lipid monomers include stearyl acrylate,hexadecyl acrylate 2,3-Bis(hexadecanoyloxy)propyl-9-methacryloyl-3,6,9-trioxanonyldimethammonium iodide,12-methacroyl-3,6,9,12-tetraoxadodecyl 3-(N,N-dioctadecyl carbamoyl)propionate, sodium2,3-bis(hexadecyloxy)propyl-12-methacroyl-3,6,9,12-tetraoxadecylphosphate,dioctadecadienoyl-bis(dihydroxyethyl) dimethylammonium bromide, or thelike (see Relevant Literature).

Of particular interest are polymers which involve surfactants and arecapable of forming lamellae, more particularly as vesicles. The vesiclesmay be small or large, generally from about 200 Å to 100μ in diameter,and may be unilamellar or multilamellar, being single or multichamber.Alternatively, the lipophilic polymers may be prepared as layers, in theform of micelles, tubes, helices, planar arrays, and linear orfilamentous structures.

The surfactant groups may be phosphatides, where at least one lipidchain will be at least 8 carbon atoms, usually at least about 12 carbonatoms and not more than about 100 carbon atoms, more usually not morethan about 36 carbon atoms. The surfactants may include phosphatides,phosphatidyl sugars, ethanolamine, choline, inositol, glycerol,alkylamines, etc. The surfactants may be fatty acids, amines, ethers,esters, alcohols, cardiolipins, lipid-nucleotides, gangliosides,cerebrosides, halogenated chains, particularly halogen of atomic numbers9 to 80, more particularly 9 to 35, glycolipids, lipoproteins,mycolates, tetraalkylamines, archeo-type lipids (dual-headed),lipopolysaccharides, or the like, where the lipid moieties will be atleast 8 carbon atoms and usually not more than about 100 carbon atoms,more usually not more than about 36 carbon atoms, primarily as a matterof convenience rather than necessity, lipids being readily available inthis carbon number range. Usually, there will be at least about 1percent of the polymeric units carrying a lipid group, more usually atleast about 5 percent and generally from about 10 to 100 percent of thepolymeric units will carry a lipid group. The lipid adjuvant will bepresent in at least about 0.01 weight percent, more usually at leastabout 1 percent and may be as high as 90 percent, usually being not morethan about 50 percent, more usually being not more than about 25 weightpercent of the lipid composition.

Polymer forming lipids may be found in Elbert et al., J. Am. Chem. Soc.(1985) 107:4134-4141, as well as other references cited previously, e.g.Frey et al., Laschewsky et al., and Bader et al., where fatty acidesters of glycerol or other polyols are linked through hydrophiliclinking groups or spacers to a polymerizable hydrophilic moiety. Thissame approach may be employed in the subject invention by combining alipophilic adjuvant with the polymerizable lipid monomer fornon-covalent association of the adjuvant with the polymer.Alternatively, the adjuvant may be modified with an acrylic group orother polymerizable group, through a hydrophilic linker, and thenincorporated covalently in the polymer and lipid layer. Other polymerssuch as polydiacetylenes may be employed (see, for example, co-pendingapplication Ser. No. 933,034 filed Nov. 20, 1986).

For enzymatic condensation polymerization, procedures such as the one ofMatsumara and Takahashi, Makromol. Chem. Rapid Commun. (1986) 7:369-373,may be employed. Hydroxyacids having lipid side groups may bepolymerized, employing such acids as 12-hydroxyoctadecanoic acid,12-hydroxy-cis-9-octadecanoic acid, 16-hydroxyhexadecanoic acid and12-hydroxydodecanoic acid. The enzyme which catalyzes the reaction isobtained from Candida rugosa and is commercially available from SigmaChemical Co., St. Louis, Mo. The resulting oligomers may be combinedwith adjuvants to form vesicles or the adjuvant may be linked through anavailable hydroxyl group to a free carboxyl group of the oligomer, usingactivating agents such as carbodiimides, e.g. dicyclohexylcarbodiimide,or carbonyl diimidazole. Also oil-in-water emulsions may be employed.The monomer hydroxy acids may also be coupled to the adjuvant beforeenzymatic polyermerization.

To reduce the risk of toxic side effects, which may result fromnon-natural polymers (Bonte et al., Biochim. Biophys. Acta ( 1987)900:1-9), it may be desirable to use biodegradable polymers as the basisfor the polymer-adjuvant backbone. One example of a biodegradablepolymeric adjuvant is prepared by incorporating at least two sulfhydrylmoieties into the adjuvant. Upon mild oxidation, the sulfhydryl groupsform intermolecular disulfide bridges which cross-link the adjuvantand/or polymerizable "filler" lipid, into linear or two-dimensionalpolymers. Approaches for preparing sulfhydryl-linked phospholipids arereported elsewhere (Bonte, et al.; Weber, et al.).

In preparing the polymerized surfactant layer, the polymerizablesurfactant, for example, is dissolved in a convenient volatile solvent,e.g. non-polar, by itself, or in combination with the adjuvant.Illustrative solvents include chloroform, hexane, isopropyl ether,methylenedichloride, benzene, ketones, etc. Individual solvents orcombinations may be employed, depending upon the nature of themonomeric-surfactant or adjuvant. Trace amounts of an organichydrophilic solvent may be employed (e.g. methanol, ethanol, dimethylsulfoxide, etc.) when necessary to solubilize a particular monomer. Theconcentration of monomeric surfactant will generally be from about 0.01to 50, more usually about 0.5 to 10 mg/ml.

Depending upon the nature of the polymerizable functionality, themonomeric surfactant may now be polymerized. Polymerization isaccomplished with the lipids suspended in an aqueous solution, dissolvedin an organic solvent, or dried into a paste form. Depending upon thedesirability for mobility of the adjuvant in the layer, thepolymerization may be carried out below the transition meltingtemperature of the lipid to provide for immobility of the acyl chains.The polymer may have as few as 3 units or may have 10⁷ or more units.Polymerization may be achieved by employing short wave ultravioletlight, e.g. below 300 nm, with diynes, usually in the range of about 230to 275 nm, X-rays, electron beams, free radicals, redox agents, or otherconvenient initiators. The time for the polymerization (irradiation, forexample, will be at least about 1 min) will usually be not more thanseveral days, frequently not more than about 6 hr, more usually not morethan about 90 min.

The lipophilic polymers and adjuvants, as appropriate, may be mixed withother lipids to form the adjuvant composition, particularly as vesicles.These lipids will be surfactants, comprising a polar or charged terminusjoined to a lipophilic chain, usually an aliphatic chain of from about 8to 100 carbon atoms or more. These filler surfactants may be monomericor polymeric. If the adjuvant and surfactant are polymeric, they may bebonded to the same or different polymer chain. The monomeric lipids mayinclude: phosphatidylethanolamine, phosphatidylcholine,phosphatidylinositols, phosphatidylglycerol and mono- ordimethylphosphatidylethanolamine, fatty acids, amines, ethers, esters,alcohols, cardiolipins, gangliosides, lipid nucleotides (Ribi et al.,Biochemistry (1987) 26:7974-7976), cerebrosides, halogenated chains,glycolipids, lipoproteins, mycolates, tetraalkylamines, archeo-typelipids, steroids, e.g. cholesterol, and any other naturally occurring,synthetic surfactants or combinations thereof. The lipids may becompositions from natural sources, such as plant, bacterial, etc.

In many instances, it may be desirable to have an entity of interestjoined to either the adjuvant, the adjuvant containing polymer or thefiller surfactant containing polymer. These additives may be involved inease of isolation of the polymeric product, e.g. liposome, targeting toa particular cell type or site, binding of the polymer to anotherpolymer, forming complexes with other entities, or the like. Thus, anyligand may be joined to the polymer, where the ligand may includenucleotides, oligonucleotides, peptides, enzymes, toxins, phosphates,saccharides, phthalocyanines, drugs (monomeric or polymeric), aminoacids, chromophores, natural ligands such as biotin, lectins,bifunctional reagents, effector molecules, sugars, antigens, dyes, crownethers, silanes, steroids, haptens, radioactively labelled moieties,chelating agents or the like.

The system allows for great flexibility in bringing together a varietyof moieties which may serve individual functions, while providing foradjuvant activity. Thus, oligonucleotides may be employed, which maythen be complexed specifically and non-covalently with large nucleicacid polymers. In this manner, monomeric units may be prepared, whichmay be non-covalently bonded to a large polymer to provide for sidechains which may be the same or different. Chromophores may be includedwhich may be used to monitor concentration, size of particles, numbersof liposomes, etc. Ligands such as biotin may be employed, which may beused for binding to avidin or streptavidin ("avidin"), for bringingtogether multiple groups, for separation or the like.

By employing various mixtures, one can modify many of the adjuvant'sproperties. By using the adjuvant, or adjuvant polymer, by itself or inconjunction with the filler surfactants, one may control morphology andmore closely mimic natural biological cell walls. In addition, bypreparing vesicles, one may encapsulate drugs within the vesicle, whichmay act to stimulate or retard cell growth. For example, one could beinterested in stimulating B-cell proliferation with mitogens, whilesuppressing T-cell types with suppressing agents. By employing cleavablelinkages, where the linkage is cleaved under physiologic conditions,including enzymatic hydrolysis, one can carry a variety of compounds,such as drugs or prodrugs, with the adjuvant polymer or the polymericfiller surfactant. These drugs may serve to selectively inhibit T-cellsas opposed to B-cells. In addition, drugs may be included in the polymerby linking to a monomer through a physiologically cleavable linkage.

It may be necessary to employ combination therapies such asimmunotherapy-chemotherapy. To this extent, the polymer-adjuvant systemmay be functionally modified to contain chemotherapeutic drugs.Alternatively, the-drugs may be encapsulated within the polymer-adjuvantvesicles. Liposomal encapsulation has been shown to reduce the toxicityof several chemotherapeutic agents. The drugs may be covalently ornon-covalently associated with monomeric or polymeric filler surfactantsor adjuvants. The mol ratios of chemotherapeutic drugs to adjuvants maybe controlled by chemical introduction of specific functional groups atdesired locations on the polymer backbone or on the monomericsurfactants. The chemotherapeutic drug may also be incorporated directlyinto the polymer backbone. For example, methacrylalannomycin, preparedas described (Molz, Ph.D. Dissertation, Mainz, West Germany (1982)), maybe co-polymerized with a methacryl-containing adjuvant or co-polymerizedwith the filler surfactant. Each configuration provides for the slowsustained release of both the adjuvant and chemotherapeutic drug.

Another approach involves the non-covalent association of achemotherapeutic drug with the polymeric adjuvant system. Adriamycin,for example, which is known to associate with cardiolipin (Goormaghtighet al., Biochemistry (1987) 26:1789-1794), may be complexed withpolymeric adjuvant liposomes containing cardiolipin.

The physical state of the polymeric adjuvant system may be modulated bychoosing surfactants (polymerizable or non-polymerizable) with theappropriate acyl chain composition and configuration. For example,butadiene, methacryloyl and sulfhydryl-linked lipids may be polymerizedand remain stable in a fluid state, whereas diacetylenic lipids requirecrystallinity for polymerization. The lipid phase state was shown to becritical for the binding of anti-lipid antibodies (Rauch et al., J.Biol. Chem. (1986) 261:9672-9677). Lateral phase separation of variousadjuvant or filler surfactant compositions may be desirable undercertain circumstances. Co-existing solid-liquid or monomer-polymerphases may play a role in controlling the proper delivery of adjuvantsinto the host. Furthermore, the dimensions and morphologies of colloidalaggregates of the lipids (e.g. tubular, helical, filamentous, hexagonalphase, uni- or multilamellar, etc.) may be controlled by varying thelipid composition.

Depending upon the nature of the composition, the subject compositionsmay be prepared in a variety of ways. Vesicles may be prepared inaccordance with conventional techniques, by combining the adjuvantpolymer by itself, or in combination with filler surfacants in anappropriate aqueous medium, subjecting the medium to agitation, e.g.sonication, or slow swelling, for sufficient time to form liposomes orother colloidal aggregates, followed by removal of the medium. Fortechniques for preparing liposomes, see, for example, U.S. Pat. Nos.4,311,712; 4,310,506; 4,302,549; 4,261,975; 4,241,046; 4,235,871; and4,299,360. For forming tubes or other structural entity, see, forexample, co-pending application Ser. No. 933,034, filed Nov. 20, 1986;and Ribi et al., Biochemistry (1987) 26:7974-7976. For forming layers,which may be mono-, bi- or higher order, see, for example, Sugi, J. Mol.Electronics (1985) 113-17; Bader et al., Adv. in Polymer Sci. (1985)64:1.

The subject adjuvants may be combined with the immunogen in aphysiologically acceptable medium in accordance with conventionaltechniques for employing adjuvants. Various media include water,phosphate buffered saline, aqueous ethanol, dimethyl sulfoxide, otherbuffers which may contain trace amounts of triethylamine or othermolecules which may aid in solubilizing the composition. Generally, theimmunogen will be present in from about 0.01 μg to 1000 μg, more usuallyfrom about 5 μg to 100 μg. The adjuvant will be present, based on theimmunogen in from about 0.1 to 100 mol ratio, more usually from about 1to 10 mol ratio.

Polymeric adjuvant systems are amenable to use in conventional adjuvantdelivery systems. While the subject invention is preferably used withoutoils, in some instances the use of oils may be acceptable. For example,polymer-adjuvants may be mixed with squalene oil in oil-in-wateremulsions (see cited literature) and administered by injection into thehost. Ultimately it may be beneficial to eliminate the use of oilscompletely.

Rather than mixing the immunogen with the adjuvant, the immunogen may becovalently or non-covalently reacted with the adjuvant. By havingreceptors for the immunogen bound to the vesicles or lipid layer, theimmunogen may become non-covalently bound. Alternatively, as describedpreviously, cross-linking or bridging agents may be employed for bondingthe immunogen to a lipid unit in the layer. When the immunogen isincluded in the medium during preparation, it may be captured in thelumen and depending on the immunogen, or may be exposed at the surfaceof the vesicle.

The resulting formulation may be administered in a single administrationor multiple administrations spaced from about 3 days to a month or moreapart. The administration may be parenteral, topical, with the aid oftransdermal patches, subcutaneous, peritoneal, intravascular, oral, byinhalation, or the like. The particular method of administration is notcritical to this invention.

The wide range of polymeric adjuvant formulations and various means ofpreparation and polymerization provide for great versatility in designand function. Several configurations of polymeric adjuvant systemsfollow: I) lipophilic adjuvants (naturally occurring, chemicallymodified, semi-synthetic, or fully synthetic), derivatized withpolymerizable moieties, are mixed with monomeric surfactants to formstable lamellar; II) polymerizable lipophilic adjuvants areco-polymerized with lipophilic surfactants; III) polymers of lipophilicadjuvants are mixed with polymers of lipophilic surfactants; IV)polymerizable lipophilic adjuvants are co-polymerized with polymerizablehydrophilic adjuvants; V) monomeric non-polymerizable lipophilicadjuvants are mixed with polymeric surfactants; VI) lipophilic polymericadjuvants of various combinations are mixed alone or in combination withmonomeric adjuvants with conventional oil-in-water emulsion systems (seerelevant literature); and various combinations thereof. Polymerizationof a particular system may be carried out with the lipids in a lamellarstate, amorphous glassy state (neat) or with the monomers solubilized inan organic solvent. Polymerization, as mentioned earlier, may beinitiated by a number of methods. A wide range of polymerizablesurfactants that may serve as fillers has been reported (Bader, H. etal., Advances in Polymer Membranes In: Polymer Membranes 64 (1985)(Gordon, M. ed.) Springer Verlag, New York; Fendler, Science (1984)223:888). In each case adjuvants are incorporated into stablepolymerized assemblies of colloidal dimensions. The polymerized adjuvantsystems may be used alone or in combination with antigen (viral,bacterial, tumor or the like) for the treatment of malignant orinfectious diseases through non-specific or specific immunotherapy.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Preparation of Lipophilic Adjuvants Linked to PolymerizableMoieties through Hydrophilic Spacers Synthesis of thepolymerizable-linker 12-methacryloyl-3,6,9,12-tetraoxadecyl-succinate(MTS)

A solution containing methacroyl chloride (1.0×10⁻² mol) and dry dioxane(30 ml) is added dropwise to a solution containing tetraethyleneglycol(2.0×10⁻² mol), diisopropylamine (1.0×10⁻² mol) and dry dioxane (30 ml)stirring at 10° C. After 30 min the reaction is warmed to 23° C. andstirred for 40 min. The solvent is removed in vacuo, the residuumresuspended in diethyl ether, extracted twice with double glassdistilled water, and then the organic phase is allowed to dry overmagnesium sulfate. The solvent is removed in vacuo and then the product,methacryloyltetraethyleneglycol, purified by reverse phase columnchromatography.

Methacryloyltetraethyleneglycol (5×10⁻³ mol), stirring at 40° C. in drydioxane (20 ml) and triethylamine (5×10⁻³ mol), is succinylated withsuccinic anhydride (5×10⁻³ mol). The solvent is removed in vacuo and theproduct, 12-methacryloyl-3,6,9,12-tetraoxadecyl-succinate (MTS), useddirectly or further purified by reverse phase column chromatography. Theproduct is characterized by Liquid Secondary Mass Spectrometry (LSIMS),H¹ -NMR, and IR.

Synthesis of 12-methacryloyl-3,6,9,12-tetraoxadecylsuccinyl-O⁶-monophosphoryl lipid A (MTS-MPL):

MTS (42 mg, 1.2×10⁻⁴ mol) is dissolved in dioxane (2 ml). After theaddition of dicyclohexylcarbodiimide (36 mg, 1.7×10⁻⁴ mol) the solutionis stirred at 40° C. for 10 min and then let stand at 23° C. for 1 hr. Aprecipitate of dicyclohexyl urea is removed by filtration through glasswool, and the filtrate added to a solution containing monophosphoryllipid A (100 mg, -5.82×10⁻⁵ mol), prepared as described (Ribi et al.,Cancer Immun. Immunotherap. (1982) 12:91-96; Qureshi et al., J. Biol.Chem. (1982) 257:11808-11815; Ribi et al., Rev. Infect. Dis. (1984)6:567-572), triethylamine (0.016 ml), and 2 ml dichloromethane. Afterstirring at 23° C. for 8 hr, half of the solvent is removed in vacuo andthe remaining mixture fractionated by liquid column chromatography(silica gel) using a chloroform:methanol:water:ammonium hydroxidegradient. The solvent is removed in vacuo and the product,12-methacryloyl-3,6,9,12-tetraoxadecylsuccinyl-O.sup. 6 -monophosphoryllipid A, is characterized by LSIMS, H¹ -NMR, and IR.

Synthesis ofN-acetylmuramyl-L-alanyl-D-isoglutamylaminocaproyl-di-(10,12-nonacosadiynoyl)-phosphatidylethanolamine(MDP-aminocaproyl-DNDPE)

t-BOC-aminocaproic acid (93 mg, 4.0×10⁻⁴) prepared as described (Moroderet al., Hoppe-Seyler's Z. Physiol. Chem. (1976) 1651-1653), anddicyclohexylcarbodiimide (167 mg, 8.1×10⁻⁴ mol) are combined in 3.0 mlof dry dichloromethane and stirred at room temperature for 40 min. Theprecipitate of dicyclohexyl urea is removed by filtration through glasswool, and the filtrate added to 104 mg (1.0×10⁻⁴ mol) ofdi(10,12-nonacosadiynoyl)-phosphatidylethanolamine (DNDPE) in 1 ml ofdry dichloromethane and 150 μl of dry pyridine. The mixture is stirredfor 2 hr and then applied to a column of silica gel. The column iswashed with chloroform:acetone (1:1 v/v) and eluted withchloroform:methanol under low pressure (10 psi). The solvent is removedin vacuo yielding 85 mg of t-BOC-aminocaproyl-DNDPE. Infrared (KBrpellet): bands at 1660 cm⁻¹ (carbonyl) and 3400 cm⁻¹ (amide).

The amino-protecting group is removed from 85 mg oft-BOC-aminocaproyl-DNDPE by stirring in a solution of 33%trifluoroacetic acid and 67% dichloromethane (5 ml) for 1-3 hr at 25° C.Solvents are removed in vacuo, and the residue fractionated on a silicagel column, yielding 60 mg of aminocaproyl-DNDPE. Digestion withphospholipase A₂ demonstrates an intact phospholipid by conversion to alysophospholipid. Infrared (KBr pellet): bands at 3350 and 3210 cm⁻¹(amine).

N-acetylmuramyl-L-alanyl-D-isoglutamine (50 mg, 1×10⁻⁴ mol) prepared asdescribed (Schwartzman et al., Preparative Biochemistry (1980)10(3):255-267), and dicyclohexylcarbodiimide (31 mg, 1.5×10⁻⁴ mol) arecombined in 1.5 ml dioxane: dichloromethane, stirred at 23° C. for 10min, and then allowed to settle for 1 hr. The precipitate ofdicyclohexyl urea is removed by filtration through glass wool, and thefiltrate is added to a solution containing aminocaproyl-DNDPE (58 mg,5×10⁻⁵ mol) triethylamine (6×10⁻⁵ mol), and 1.5 ml dioxanedichloromethane. After 6 hr. the reaction mixture is purified by liquidcolumn chromatography (silica gel) using a chloroform:methanol gradientunder low pressure (10 psi). The solvent is removed in vacuo and theproduct, MDP-aminocaproyl-DNDPE, characterized by phospholipase A₂digestion, LSIMS, H¹ -NMR, and IR.

Synthesis ofmono(12-methacyloyl-3,6,9,12-tetraoxadecylsuccinyl)-trehalose dimycolate(MTS-TDM)

MTS (0.1 mmol), ethylchloroformate (0.11 mmol), and triethylamine (0.11mmol) are combined with dry dioxane (2.5 ml) and stirred at 23° C. After1.5 hr the mixture is added dropwise to a stirring solution of TDM (0.1mmol) prepared as described (Toubiana et al., Carbohdr. Res. (1975)44:308; Azuma et al., J. Natl. Cancer Inst. (1974) 52:95-101; and Promeet al., Eur. J. Biochem. (1976) 63:543), and dry dichloromethane (10ml). After 24 hr the solvent is removed in vacuo and the productsseparated by liquid column chromatography using achloroform:methanol:water gradient as the eluent. Fractions containingonly a single spot by thin-layer chromatography are dried in vacuo andcharacterized by LSIMS, H¹ -NMR, and IR. Only the mono-MTS substitutedTDM, mono(12-methacryloyl-3,6,9,12-tetraoxadecylsuccinyl)-trehalosedimycolate (MTS-TDM), is used for polymerized adjuvant systems below.

Polymerized Adjuvant Formulations

Formulation I: Polymerized vesicles containing MDP-aminocaproyl-DNDPE(0.01 mmol) and L-α-distearoylphosphatidylcholine (0.003 mmol) areprepared by methods described (Hub et al., Angew. Chem. Int. Ed. Engl.(1980) 19:938; Johnston et al., Biochem. Biophys. Acta (1980) 602:57;and Lopez et al., J. Am. Chem. Soc. (1982) 104:305), with modifications.The lipids are dissolved in chloroform:methanol (10 ml, 3:1 v/v) anddried into a thin film on the bottom of a round-bottom flask (50 ml) byrotoevaporation. The lipids are further dried in vacuo for 1 hr. Wateror buffer (e.g. phosphate buffered saline) is added to bring the finalconcentration of lipid/water to 5-10 mg/ml. Vesicles are formed asdescribed (see relevant literature above) by slowly warming the flask toa temperature greater than the melting transition of the lipids (>50°C.) for 1-2 hr, followed by gently stirring the solution, and thenlowering the temperature below the melting transitions of the lipids(less than 20° C.). Polymerization is performed as described (seerelevant literature). Vesicles are characterized by electron microscopy,light scattering, and spectroscopic analysis. For the enhancement ofantibody formation (see Biological Assays), antigen (1.5 parts by weightantigen to total weight of polymer prepared above) is added prior topolymerization in one experiment and after polymerization in anotherexperiment.

Formulation II: Polymerized vesicles containing co-polymerizedpolyMTS-MPL (0.01 mmol based on monomer), polyMTS-TDM (0.01 mmol basedon monomer), polysodium2,3-bis(hexadecyloxy)-propyl-12-methacryloyl-3,6,9,12-tetraoxadecylphosphate(0.05 mmol based on monomer) prepared as described by Elbert et al.(1985), and poly-2-hydroxyacrylate (0.15-0.3 mmol) are prepared bymethods described (Elbert et al. (1985) supra; and Laschewsky et al.(1986) supra) with modifications. The lipids and 2-hydroxyacrylate aredissolved in toluene (10 mg/ml final concentration) by warming andsonication. 2,2'-azoisobutyronitrile is added (0.01-0.2 mg/ml finalconc.), the mixture flushed with nitrogen, and then polymerization iscarried out at 60° C. for 20 hr. Polymers are precipitated withmethanol:acetone, rinsed with methanol:acetone, and then liposomesprepared by the addition of water or buffer (1-5 mg/ml finalconcentration) and ultrasonication at 60° C. for 0.5 hr. Polymerizedvesicles are characterized by electron microscopy, light scattering, andspectroscopic analysis. For the enhancement of antibody formation (seeBiological Assays), antigen (1.5 parts by weight antigen to the totalweight of adjuvant components prepared above) is added directly prior tosonication in one experiment and after sonication in another experiment.

Formulation III: Precipitated polymers of polyMDP-aminocaproyl-DNDPE(0.01 mmol based on monomer, prepared from polymerized vesicles of pureMDP-aminocaproyl-DNDPE as described in formulation I), polyMTS-TDM (0.01mmol based on monomer, prepared as described in formulation II),poly-2,3-bis(hexadecyloxy)propyl-12-methacryloyl-3,6,9,12-tetraoxadecylsuccinate(0.05 mmol based on monomer, prepared as described by Elbert et al.(1985)), and polyMTS-MPL (0.01 mmol based on monomer, prepared as informulation II) are gently ground to an oily powder. Liposomes areprepared by the addition of water or buffer (1-5 mg/ml finalconcentration) and ultrasonication at 60° C. for 1 hr. Vesicles arecharacterized by electron microscopy, light scattering, andspectroscopic analysis. For the enhancement of antibody formation (seeBiological Assays), antigen (1.5 parts by weight antigen to the totalweight of adjuvant component prepared above) is added directly prior tovesicle formation in one experiment and after vesicle formation in asecond experiment.

Formulation IV: Polymerized vesicles containing co-polymerized,polyMTS-MPL (0.01 mmol based on monomer), polyMTS-TDM (0.01 mmol basedon monomer), polyN¹ -(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N⁶-acryloylhexamethylenediamine (0.01 mmol, based on monomer, prepared asdescribed by Kohorlin and Abashev, Bioorg. Khim. (1984)10(8):1119-1126), and acrylamide (0.1-0.5 mmol based on monomer) areprepared as in formulation II. For the enhancement of antibody formation(see Biological Assays), antigen (1.5 parts by weight antigen to totalweight of adjuvant component prepared above) is added to the mixture ofpolymers directly prior to vesicle formation in one experiment and aftervesicle formation in another experiment.

Formulation V: TDM (0.01 mmol, from sources cited in Synthesis ofMTS-TDM), MPL (0.01 mmol, from sources cited in Synthesis of MTS-MPL),MDP-aminocaproyl-DNDPE (0.01 mmol, prepared as described earlier), andpoly(12-methacryloyl-3,6,9,12-tetraoxadecyl)-3-(N,N-dioctadecylcarbamoyl)-propionate(0.05-0.1 mmol based on monomer, prepared and polymerized as describedby Elbert et al. (1985) supra) are dissolved in chloroform (25 ml) anddried into a thin film in the bottom of a round-bottom flask byrotoevaporation. The lipids are further dried in vacuo for 1 hr, andthen slowly hydrated with water or buffer (5-10 mg/ml finalconcentration) at 70° C. for 1-2 hr. If any flocculent matter remains,the solution is further dispersed by sonication (1-30 min, 50° C.). Forthe enhancement of antibody formation (see Biological Assays), antigen(1.5 parts by weight antigen to the combined weights of TDM, MPL, andMDP-aminocaproyl-DNDPE as combined above) is added with the water orbuffer prior to warming in one experiment and after warming in anotherexperiment.

Formulation VI: Liposomes containing TDM (0.01 mmol, from sources citedin Synthesis of MTS-TDM), MPL (0.01 mmol, from sources cited inSynthesis of MTS-MPL), MDP-aminocaproyl-DNDPE (0.01 mmol, prepared asdescribed earlier), anddimethyl-bis(2-octadeca-2,3-dienoyloxyethyl)ammonium bromide (0.03 mmol,prepared as described by Gaub et al., Biophysics J. (1984) 45:725-731)are prepared as in formulation V with modifications. In one experiment,the vesicles are formed by hydration (5-10 mg total lipid per ml water)at 60° C. as described. After 1-2 hr the solution is gently stirred for10 min, the temperature of the solution lowered to 15° C., the solutiontransferred to a quartz vial, and then polymerized by irradiation (254nm) with a Pen-Ray-UV-Lamp (A.R. Vetter Co., Rebersburg, Pa.) for 1 hr.In a similar experiment, polymerization is carried out at 35° C.Vesicles are characterized by electron microscopy, light scattering, andspectroscopic analysis. For enhancement of antibody formation (seeBiological Assays), antigen (1.5 parts by weight antigen to the combinedweight of TDM, MPL, and MDP-aminocaproyl-DNDPE as prepared above) isadded, in each experiment, to the lipid composition prior to hydration.

Formulation VII: The aqueous preparations of adjuvant formulations I-VI,prepared with or without antigen (see respective formulations) arelyophilized to yield a paste-like powder. The powder (10 mg) is combinedwith squalene oil (80 μl) in the bottom of a dry glass tissue grindertube (30 ml) and then ground with a snug-fitting teflon pestle (1000 rpmfor 3 min) at 23° C. After grinding, 4 ml phosphate buffered saline,containing 0.2% Tween-80, is added and the polymerizedadjuvant/oil/water mixture emulsified by further grinding (1000 rpm, 4min). Aliquots of emulsion are vortexed and then used immediately (seeBiological Assays).

Biological Assays

I. Enhancement of Resistance to Viral Infection. For protection againsta mouse virulent strain of influenza A virus (A/PR/8/34(HINI)),polymerized adjuvant formulations I-VII are injected intravenously intoC57BL/10 X BALB/c mice via the method described by Mashihi et al., Int.J. Immunopharmac. (1986) 8:(3):339-345. In one experiment an aliquot ofan adjuvant formulation (containing 450 μg adjuvant based on the totalmonomer composition of the adjuvants present) is injected intravenouslyinto a mouse. After three weeks the mouse is infected by an aerosolspray of the virus (A/PR/8/84). Lung virus titres are determined 72 hrafter the infection, as described by Mashihi et al. (above). In a secondexperiment, viral protein subunits (A/Victoria/3/75 and B/HongKong/5/72) are injected as antigen (4 μg per dose) intramuscularly incombination with a subject adjuvant formulation (total 300 μg adjuvant,based on the total monomeric adjuvant composition per dose). The miceare infected and assayed as described above.

II. Tumor Regression. Strain 2 guinea pigs bearing 6-7 day old line-10tumors 8-10 mm in diameter (a transplantable hepatocellular carcinoma,see Rapp, Isr. J. Med. Sci. (1973) 9:366) are inoculated (intravenouslyin one experiment and intralesionally in another) with doses of adjuvantformulations I-VII (aqueous aliquots without antigen, 300 μg totaladjuvant by weight, based on total of monomer adjuvant composition perdose). Animal cure rates are assessed according to Ribi et al., CancerImmunol. Immunother. (1978) 3:171-177. Animals are considered cured whenthe tumors completely disappear, metastases are not palpable at thefirst draining lymph node, and the guinea pigs reject a rechallenge ofline-10 tumor transplantation.

III. Enhancement of Antibody Formation. Aliquots of an adjuvantformulation, I-VII (0.05-0.1 ml), containing 200 μg total adjuvant(based on total weight of monomer adjuvant in the composition), andviral antigen (10 μg, A/Victoria/3/75), are combined as described (seeFormulation), and then injected subcutaneously into C57BL/10 X BALB/cmice. Antibody titres, to the viral subunits, are measured with thestandard enzyme-linked immunosorbant assay (ELISA) after 14 and 31 days.In another experiment, compositions, identical to those described above,are injected intravenously, and then antibody titres measured after 14and 31 days. Control experiments are performed in an identical mannerbut with no adjuvant present.

In accordance with the subject invention greatly enhanced immuneresponses can be achieved. By employing adjuvants in conjunction with apolymer, the adjuvant provides a simulation of a cell, which mimics theimmune system activating effect of a microorganism. Different adjuvantscan be employed for ease of preparation, compatibility with the antigenand degree of enhancement of immune response.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

What is claimed is:
 1. A polymeric immunological adjuvant compositioncomprising:a copolymer comprising at least one adjuvant joined to awater soluble addition polymerizable acrylyl or methacrylyl monomerthrough a linking group and a lamellar forming lipid comprising a chainof from 8 to not more than 36 carbon atoms and joined to a water solubleaddition polymerizable acrylyl or methacrylyl monomer; said lipidadjuvant selected from the group consisting of monophosphoryl lipid A,lipopolysaccharide, BCG cell wall skeleton, diphosphoryl lipid A,muramyl di- or tripeptide, muramyl dipeptide-phosphatidyl-ethanolamine,muramyltripeptide phosphatidylethanolamine, trehalose monomycolate,trehalose dimycolate, lipid X, isoprinosine, and lithosperman (A, B orC).
 2. A polymeric immunological adjuvant composition according to claim1 comprising a water soluble addition polymerizable monomer free of alipid group.
 3. A combination composition comprising a polymericimmunological adjuvant composition according to claim 1in combinationwith monomeric or polymeric lamellar forming lipid molecules.
 4. Acombination adjuvant composition comprising:monomeric lamellar formingadjuvant, wherein said adjuvant is lamellar forming or is joined to alamellar forming lipid, and a polymeric immunological adjuvantcomposition according to claim 1.